Organic solutes rescue the functional defect in ΔF508 cystic fibrosis transmembrane conductance regulator
Organic solutes rescue the functional defect in ΔF508 cystic fibrosis transmembrane conductance regulator
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DOI：
10.1074/jbc.m309076200
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发表时间：
2003-12-19
影响因子：
4.8
通讯作者：
Guggino, SE
中科院分区：
文献类型：
--
作者：
Zhang, XM;Wang, XT;Guggino, SE
囊性纤维化中最常见的缺陷是囊性纤维化跨膜传导调节因子（DeltaF 508 CFTR）的508位苯丙氨酸缺失，这减少了该蛋白质向细胞表面膜的运输。先前的研究表明，低温和高浓度的甘油或N-氧化三甲胺可以部分抵消DeltaF 508 CFTR的加工缺陷。本研究调查是否生理相关浓度的有机溶质，累积的协同转运蛋白，可以拯救DeltaF 508 CFTR的错误处理。在稳定表达DeltaF 508 CFTR的IB 3细胞或NIH 3 T3细胞中，单独的肌醇或依次给予肌醇、甜菜碱和牛磺酸增加了核心糖基化、内质网停滞的DeltaF 508 CFTR向完全糖基化形式的CFTR的加工。使用瞬时转染的COS 7细胞的脉冲追踪实验表明，有机溶质也增加了核心糖基化形式的绿色荧光蛋白-DeltaF 508 CFTR的加工。此外，GFP-DeltaF 508 CFTR的复合糖基化形式的半衰期延长表明这种处理稳定了蛋白质的成熟形式。纯化的NBD 1稳定性和聚集的体外研究表明，肌醇稳定了DeltaF 508和野生型CFTR，并抑制了DeltaF 508的错误折叠。最重要的是，用这些可转运的有机溶质处理CF支气管气道细胞恢复了细胞表面膜中CFTR和向外整流氯离子通道的cAMP刺激的单通道活性，还恢复了毛喉素刺激的宏观Cl-36（-）流出。我们得出结论，有机溶质可以通过稳定DeltaF 508 CFTR的复合糖基化形式来增强DeltaF 508 CFTR对质膜的加工来修复CFTR功能。
The most common defect in cystic fibrosis, deletion of phenylalanine from position 508 of the cystic fibrosis transmembrane conductance regulator (DeltaF508 CFTR), decreases the trafficking of this protein to the cell surface membrane. Previous studies have shown that low temperature and high concentrations of glycerol or trimethylamine N-oxide can partially counteract the processing defect of DeltaF508 CFTR. The present study investigates whether physiologically relevant concentrations of organic solutes, accumulated by cotransporter proteins, can rescue the misprocessing of DeltaF508 CFTR. Myoinositol alone or myoinositol, betaine, and taurine given sequentially increased the processing of core-glycosylated, endoplasmic reticulum-arrested DeltaF508 CFTR into the fully glycosylated form of CFTR in IB3 cells or NIH 3T3 cells stably expressing DeltaF508 CFTR. Pulse-chase experiments using transiently transfected COS7 cells demonstrated that organic solutes also increased the processing of the core-glycosylated form of green fluorescent protein-DeltaF508 CFTR. Moreover, the prolonged half-life of the complex-glycosylated form of GFP-DeltaF508 CFTR suggests that this treatment stabilized the mature form of the protein. In vitro studies of purified NBD1 stability and aggregation showed that myoinositol stabilized both the DeltaF508 and wild type CFTR and inhibited DeltaF508 misfolding. Most significantly, treatment of CF bronchial airway cells with these transportable organic solutes restores cAMP-stimulated single channel activity of both CFTR and outwardly rectifying chloride channel in the cell surface membrane and also restores a forskolin-stimulated macroscopic Cl-36(-) efflux. We conclude that organic solutes can repair CFTR functions by enhancing the processing of DeltaF508 CFTR to the plasma membrane by stabilizing the complex-glycosylated form of DeltaF508 CFTR.